DNA methylation is a reaction occurring in “cytosine”, which is one of the bases constituting DNA. It controls when and from which gene a protein should be produced and also correctly regulates a step of transcription into RNA that is required for protein synthesis. The methylation of cytosine (production of 5-methylcytosine) plays a role of inactivating a gene function, for example, without depending on a nucleotide sequence of the gene. It is known that even cancer might be caused when an abnormality occurs in methylation. From the viewpoint that the regulatory mechanism of phenotype expression is affected not by a nucleotide sequence but by an acquired function, i.e., the viewpoint of epigenetics, cytosine methylation is a very important phenomenon.
In this connection, it is necessary to measure immediately a position in DNA at which methylation occurs and evaluate whether the amount of the methylation is normal or not. In particular, when DNA methylation is measured in the field of medical therapeutics, rapid and accurate obtainment of measurement results is required.
As a conventional method for detecting methylcytosine (5-methylcytosine), a method based on treatment with metabisulfite salt is known. According to the method based on treatment with metabisulfite salt, DNA as a test sample is treated with metabisulfite salt and subjected to PCR and sequencing process. As a result, the methylated cytosine and non-methylated cytosine are detected as cytosine and thymine, respectively. The detection based on treatment with metabisulfite salt is disclosed, for example, in Patent Document 1 (Japanese Patent Application National Publication (Laid-Open) No. 2004-511235). However, the treatment with metabisulfite salt is disadvantageous in that a long period of time is required for heating reaction and a non-specific damage occurs in most of genome samples so that an error is produced due to the damage (erroneous detection).
As a conventional method for detecting methylcytosine, in addition to the method based on treatment with metabisulfite salt, a method using a restriction enzyme which is sensitive to methylcytosine and a restriction enzyme which is insensitive to methylcytosine has been known (MIAMI method). According to this method, a restriction enzyme which is sensitive to methylcytosine and a restriction enzyme which is insensitive to methylcytosine are used separately, and the detection is carried out by using PCR, etc. However, as being based on an enzyme reaction, it is disadvantageous in that operational process is cumbersome and it takes several days to accomplish the detection.
Patent Document 1: Japanese Patent Application National Publication (Laid-Open) No. 2004-511235